Tim-3 engagement induces IκBα degradation and ERK activation. (A-B) Purified PB NK cells were cross-linked with plate-bound anti–Tim-3 mAb for the indicated times. Cells were stimulated with phorbol 12-myristate 13 acetate (PMA) for 15 minutes as a positive control. (A) Western blot analysis of ERK phosphorylation. Scanning densitometry was used to determine the relative expression levels of protein after normalizing to β-actin as a loading control. (B) Western blot analysis of total IκBα degradation. Data in panels A and B are representative of 4 independent experiments.